CLONING AND CHARACTERIZATION OF 4 GENES OF RHIZOBIUM-LEGUMINOSARUM BVTRIFOLII INVOLVED IN EXOPOLYSACCHARIDE PRODUCTION AND NODULATION

Four different genes of Rhizobium leguminosarum by. trifolii strain RB L5599 involved in exopolysaccharide (EPS) production were identified b y complementation of Tn5-induced EPS-deficient mutants (Exo mutants) w ith a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum by. viciae VF39 and highly homologous to a family of glycosyl transferases, two p ssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively, The three other genes were found to be closely linked on a different complementing cos mid, pssC revealed similarity to exoM and exoW of R. meliloti, both en coding glucosyl transferases involved in the synthesis of succinoglyca n. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC , consisting of two overlapping genes, pssD and pssE, that is essentia l for EPS production, Homology of pssD and pssE was found with cpsIcCF and cps14G of Streptococcus pneumoniae, respectively: two genes respo nsible for the second step in capsule polysaccharide synthesis. Furthe rmore, pssD and pssE were homologous to the 5' and 3' parts, respectiv ely, of spsK of Sphingomonas S88, which encodes a putative glycosyl tr ansferase, Structural analysis of EPS produced by fro mutants exo2, ex o4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.